Many economic and scientific interests are tied to the genetic engineering of plants. Several methods of introducing foreign genetic material such as DNA into plants, i.e., transforming plants, have evolved and are currently in use. The most common are Agrobacterium-mediated gene delivery, microprojectile bombardment and free DNA delivery to protoplasts.
DNA delivery procedures via Agrobacterium, microprojectile bombardment and direct uptake into protoplasts systems require that the transgene be under control of promoter and transcription termination signals that function in plants. The transgene sequence(s) must conform to plant consensus codon usage. Both microprojectile bombardment and protoplast systems (collectively referred to as direct DNA delivery procedures) most commonly introduce transgene sequences carried on high copy number E. coli plasmids which are easy to produce in large amounts. Agrobacterium-based DNA delivery requires that the transgene of interest be inserted into the T-DNA between the right and left border sequences on either disarmed Ti or Ri plasmids, or binary vectors followed by reintroduction into the appropriate Agrobacterium strain.
Agrobacterium-mediated transformation is currently the most widely used commercial technique. It requires the production of recombinant Agrobactrium containing the gene of interest. Transformation is then achieved by co-cultivation of the recombinant Agrobactrium with the mesophyll cells of leaf disks. The leaf disk is routinely used for dicots such as tobacco, soybean and cotton, but has not been found to be effective with monocots. Songstad, D. D. et al., Plant Cell Tissue and Organ Culture 40:1-15 (1995).
Although widely used, there are a number of drawbacks to the Agrobacterium method. It is time consuming, requiring the production and isolation of a recombinant Agrobacterium, a two day co-cultivation period, and regeneration of a plant from a single cultured cell. The method also requires the use of leaf disks which are prepared by enzymatic digestion of plant cells to break down the cell wall. After transformation, a single, transformed cell is selected and grown on antibiotic-containing media to form the new plant. The whole procedure must be carried out using sterile procedures and facilities. In addition, the method involves prolonged tissue culture and requires the capability to regenerate the cultured cells to whole plants.
Transformation of both monocots and dicots can be achieved by particle bombardment. The DNA to be delivered is attached to microparticles such as gold or tungsten beads. The beads are then “shot” into cells using a gun, usually powered by compressed air. This method has been used to transform monocots such as corn, wheat and rice that were shown to be unresponsive to Agrobacterium-mediated transformation. Songstad, D. D. et al., Plant Cell Tissue and Organ culture 40:1-15 (1995). Particle bombardment however, shares many of the same drawbacks as the Agrobacterium method. For example, the entire procedure must be carried out under sterile conditions and individual cells, not seedlings, are transformed. An additional drawback is the expense of the equipment necessary to deliver the DNA-coated microparticles to the cell (e.g., a “gene gun”).
Protoplast-mediated transformation is an alternative method for producing recombinant monocot and dicot plants. As with Agrobacterium-mediated transformation, cultured mesophyll protoplasts are transformed by introducing foreign DNA. However in protoplast-mediated transformation, the DNA is introduced directly into the cell. Direct delivery of the DNA is achieved by either chemical treatment of the protoplasts to make the cell membranes porous to the DNA or by electroporation. Songstad, D. D. et al., Plant Cell Tissue and Organ Culture 40:1-15 (1995); Morikawa, H. et al., Gene. 41:121-124 (1986). This method however, shares the drawbacks set forth above for the other methods. In addition, if electroporation is used for DNA delivery, the resulting transformed plants are often sterile.
Recent advances in plant transformation have led to the use of an electrophoretic method for the transformation of plant embryos. The embryos are either removed from the seed, or the seed is dehusked prior to electrophoresis. A cathode is then placed in direct contact with the embryo or seed and a DNA-containing medium is placed between the anode and the embryo or seed. Application of current causes the DNA to migrate from the medium into the embryo or seed cells. The embryos or seeds are then germinated. Barley and corn embryos as well as orchid protocorms have been transformed by this method. Ahokas, H., Theor. Appl. Genet. 77:469-472 (1989); Murry, L. E. et al., Biotechnology in Agriculture and Forestry 25:253-261 (1994); Griesbach, R. J. et al., Acta. Hort. 336:165-169 (1993). An advantage of this method is that it does not require sterile conditions. However, barley embryos transformed by this method produced sterile plants. Ahokas, H., Theor. Appl. Genet. 77:469-472 (1989). The method is also time-consuming, wherein the embryos must either be removed from the seeds or the seeds must be treated, i.e. dehusked, before transformation. This method also requires large amounts of plasmid DNA (approximately 10 μg). Moreover, most zygotic embryos are sensitive to the amount of current applied and often will not germinate after treatment.
It would thus be desirable to provide an improved method for transforming plants. It would be desirable for the improved method to be applicable in both dicots and monocots. It would also be desirable to provide a method for transforming plants that may be performed under normal (i.e., non-sterile) conditions with little or no preparation of the plant material. It would further be desirable to provide a method for transforming plants that produces non-sterile transformants, so that the progeny of the transformed plants also carry the transgene. It would also be desirable to provide a method for transforming plants that is commercially attractive, e.g., allowing transformation of a large number of plants at one time with high yields. It would further be desirable to provide a method for transforming plants wherein basic materials and equipment are employed so that the method is cost-effective.